The blood-brain barrier (BBB) is comprised of brain microvascular endothelial cells where tight junction protein complexes form in the intercellular space, creating a highly restrictive interface between the brain and vascular system. In oxidative stress, an excess of reactive oxygen species relative to antioxidants damages tight junction proteins, exacerbating ischemic stroke, traumatic brain injury, and Alzheimer’s disease. Previous studies revealed that poloxamer 188 (P188), a commercial triblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), mitigated BBB damage in mouse models of injuries related to oxidative stress. Given the experimental limitations of acellular and in vivo models used in prior work, there is minimal understanding of how amphiphilic block copolymers intervene in the progression of oxidative stress to stabilize the damaged BBB. To develop a platform that supports investigation of the therapeutic mechanism of amphiphilic block copolymers, we differentiated human induced pluripotent stem cells into brain microvascular endothelial cells (iBMECs) that exhibit key attributes of the human BBB. We induced oxidative stress by exposing iBMECs to hydrogen peroxide, resulting in a 30% reduction in barrier function, as indicated by the transendothelial electrical resistance, and decreased continuity of tight junction proteins claudin-5 and occludin, as quantified by analysis of confocal fluorescence microscopy images. The iBMECs under oxidative stress were treated with P188, resulting in a 10% increase in barrier function, affirming previous in vivo results. To identify improved therapeutic molecules while elucidating the relationship between polymer composition and therapeutic function, we synthesized PEO-PPO diblock copolymers with varying parameters (i.e., block length and end group functionality). The three resulting molecules granted a 20-25% increase in barrier function relative to the untreated iBMECs under oxidative stress. In this work, we established a human-based in vitro model of the BBB under oxidative stress that reports damage and supports the evaluation of block copolymers. This tissue engineered model of the BBB is highly tunable (i.e., can include physiological flow and co-culture with other cell types in the neurovascular unit), enabling deeper investigation into the mechanism of polymer-mediated barrier stabilization while continuing to bridge an unfilled gap between acellular and in vivo models.

Samira Azarin

University of Minnesota

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